All viruses depend on their host cells for replication. However, many aspects of virus-host cell interaction - such as the role of membrane trafficking in (+) RNA virus replication - are still poorly understood, in part due to a lack of sufficiently precise and powerful tools. This Exploratory and Developmental Research Proposal explores a new strategy to identify virus-host cell interaction by employing a large collection of bacterial effector proteins as cell biological probes, based on the observation that these proteins frequently target the same cellular pathways used by (+) RNA viruses. Because bacterial effector proteins are genetically encoded enzymes that have evolved to manipulate host cell biology with high specificity and activity, they have distinct advantages over other reagents for probing virus-host interaction, such as RNAi or pharmacological agents. By using a high-throughput screening approach, we will express a panel of 435 bacterial effector proteins to manipulate host cell biology, and examine how these perturbations affect the replication of hepatitis C virus, dengue virus, and yellow fever virus, three representative members of the Flaviviridae. Hits will then be prioritized for further mechanistic characterization and follow up studies. Our preliminary data demonstrate feasibility of this approach and provide interesting leads for future experiments. The outcome of these studies will provide proof of concept for our strategy, will generate a set of unique tools to study an important group of (+) RNA viruses, and will collaboratively synergize with ongoing research into bacterial effector mechanisms. We envision a future experimental niche wherein virologists and cell biologists will be able to utiliz a toolkit of bacterial effector proteins to probe many different biological questions.